IgY derived from hyperimmunized chicken eggs has been added as a supplement to animal feed to provide passive immunity in other animals. By a process of vaccination, chickens or other egg-laying animals can be immunized against specific microorganisms and other antigens. Increased titers of antibodies can be obtained by a process of hyperimmunization. Hyperimmunization procedures are well-known in the art and have been described in detail. High amounts of specific antibodies can be obtained by immunizing animals with specific antigens and isolating the antibodies from the egg yolk, milk, colostrum or serum. While it has been known since 1893 that the IgY antibody is found in the yolks of chicken eggs (Klempeler 1893), it is only in the last 20 years or so that use of IgY has gained ground as a practical source of passive immunity in livestock or other animals. While nature intended liquid colostrum as the source of antibodies to provide the initial passive immunity that a newborn animal receives from its mother, in the modern agriculture industrial practice it has become necessary to provide passive immunity from various antibody supplements in order to achieve profitability.
The supplements that the animal feed industry uses to provide passive immunity to animals have been derived from colostrum, blood serum and, more recently, from chicken egg IgY antibodies. The use of IgY antibodies to provide passive immunity provides certain advantages over the use of antibodies found in colostrum or antibodies found in blood serum. These advantages include the relative cost efficiency to create specifically directed antibodies against pathogenic organisms compared to colostrum or blood serum antibodies.
Passive immunity is the transfer of active humoral immunity in the form of ready-made antibodies, from one individual to another. Passive immunity can occur naturally, when maternal antibodies are transferred to the offspring. It can also be induced artificially, when high levels of antibodies specific for a pathogen or a toxin are recovered from immunized individuals and administered to non-immune individuals. The antibodies' transfer may be carried out via systemic, intravenous or oral routes. The oral route is the route of choice for localized treatment of digestive tract infections. Immunity derived from passive immunization lasts for only a short period of time, i.e., for as long as the antibodies remain in the organism, but it provides immediate protection.
In animals, the administration of preformed, specific antibodies is an attractive approach to establish protective immunity against viral and bacterial pathogens. It is becoming a more interesting alternative to control the increasing number of antibiotic-resistant organisms. Passive immunization can also be used against organisms that are non-responsive to antibiotic therapy.
Antigen-specific IgY antibody can be produced on a large-scale from eggs laid by chickens immunized with selected antigens (Hatta et al., 1997). The laying hen transfers all antibody isotypes found in the chicken to the egg, i.e., IgY, IgM, and IgA antibodies. The yolk contains only IgY, while IgM and IgA are found only in the white. The chicken's serum IgY level is reflected in the egg yolk shortly after a single administration of antibody, about one week. Egg yolk contains approximately 3-25 mg IgY/ml. Depending on its weight, therefore, each egg could provide 40-500 mg IgY.
The use of IgY for passive immunization has been studied extensively, demonstrating its effectiveness in preventing or treating infectious diseases caused by various pathogens in animal models, especially those of the gastrointestinal (GI) tract. Antibodies are usually administered in the feed in several forms: whole egg powder, whole yolk powder, water-soluble fraction powder or purified IgY material. The powdered egg formats containing IgY or other anti-bacterial components are typically packaged into containers or made into pastes. The powders are mixed into dry feed and subsequently given to the animal. The pastes are squirted into the mouth of the animal.
The animal feed industry also uses liquid feed supplements as a standard method of providing supplements to animals, due to cost considerations and ease of use considerations. A liquid format is desirable since it is easy to administer liquids in drenches or even in watering systems. However, a stabilized liquid format that maintains titers of IgY derived from hyperimmunized chicken eggs for an extended shelf life has not been developed.
The methods that are currently used to preserve IgY titer from eggs involve either expensive spray-drying or freeze-drying techniques for the whole egg or egg yolk and converting the egg into a powder. However, both of these methods have problems in maintaining the original structure of the IgY and binding activity of the IgY molecule. Spray-drying methods use heat during the processing of the egg material that will denature the IgY molecule to a large extent. IgY denatures when thermally treated at temperatures higher than 75° C. (Chang et al., 1999). Freeze-drying methods involve freezing the egg material which also can structurally alter the IgY molecule. Chansarkar (1998) showed that frozen or freeze-dried IgY resulted in loss of antigen-binding activity and a significant drop in the solubility under the conditions of high salt and protein concentrations.
Keeping the IgY antibody in a liquid state from the time it is in the egg to the time it is used is the best method to maintain the structure of the IgY molecule, to prevent degradation, and preserve its binding activity in providing passive immunity. Keeping the hyperimmune eggs in their egg shell without cracking them open is not a practical commercial method. Whole eggs stored at room temperature will naturally start degrading within several weeks and are also not practical to use as a delivery means to the animal.
Keeping whole egg or yolk products in a stable liquid state at room temperature without degradation of the liquid matrix or the IgY content for periods of time greater than few hours or even a day is a problem to the animal feed industry. This is due to oxidation issues, contamination issues, and coagulation issues of liquid egg products. Attempts at keeping whole eggs or liquid yolks as a stabilized liquid matrix have been the subject of a number of patents for human products. These methods principally involve pasteurization of the egg product at various temperatures. For example, U.S. Pat. No. 6,403,141 is directed to a method of obtaining a long shelf life for liquid egg products using pasteurization techniques. This method allows storage of whole egg products at room temperature for 3 months without the incorporation of additives. However, this patent has not addressed the overriding issue of IgY stability when storing the products at room temperature and use a different technology approach than the current invention to extending shelf life of the matrix.
Other methods of purifying IgY on a commercial scale may lose important factors that may be involved in the efficacy of whole chicken eggs or whole yolks when providing antibacterial properties. While IgY is the focus of passive immunity, it has also has been shown that anti-bacterial properties were associated with egg yolk components other than the IgY. Among these components are the granule high-density lipoprotein (HDL) (Kassaify et al., 2005), the plasma low-density lipoprotein (LDL) (Brady et al., 2002), and the egg-yolk-derived sialyloligosaccharide (YDS) (Sugita-Konish et al., 2002). As such, it desirable to maintain these components when providing passive immunity in a liquid egg product by making sure the entire yolk is used or the entire contents of the egg are used and not using just purified IgY.